All blood cells are derived from rare hematopoietic stem cells (HSCs) that reside in the bone marrow in the adult. Several transcription factors are critical for the formation and subsequent function of HSCs, and for the development of specific blood lineages. Study of the molecular mechanisms underlying blood cell specification and function has contributed immeasurably to our understanding of mammalian development, stem cell biology, and hematopoietic malignancies. The Growth Factor Independent (Gfi)-genes, Gfi-1 and Gfi-1b, are essential for various aspects of blood cell development and function. Gfi-1 is required for functional integrity of HSCs; without Gfi-1, HSCs proliferate excessively and lack self-renewal activity. Subsequently, Gfi-1 is important for the choice between neutrophilic and monocytic lineages, differentiation of neutrophils, and also for proper lymphoid development. Gfi-1b is required for normal erythroid and megakaryocytic cell maturation. Although much has been learned, important questions remain unresolved. The overall goal of this proposal is to define the mechanisms by which the Gfi proteins function in both HSCs and specific lineages, as this will be the most direct route to identifying the pathways used to sustain HSCs and also promote cellular maturation, rather than malignant transformation. The principles gained from the proposed studies should be readily applicable to other nuclear regulators central to the blood system and to factors employed in other organ systems. Aim 1 focuses on genetic experiments designed to determine the contribution of Gfi-1b to the formation and/or function of HSCs. Conditional knockouts of Gfi-1 and Gfi-1b in the mouse will be combined, and the effects of combined gene loss on development of the hematopoietic system and on the survival, proliferation, and self-renewal of HSCs in the adult will be determined. A systematic genomic approach will be developed to identify the critical gene targets regulated by the Gfi proteins in HSCs. Aim 2 centers on biochemical mechanisms of transcriptional repression by the Gfi proteins. Work in this laboratory has identified a repressor complex recruited to gene targets by the Gfi proteins. This complex includes the histone demethylase LSD1. Further work will be directed to elucidation of the details of the action of the repressor complex at target genes, and the roles of LSD1 in HSCs and specific hematopoietic lineages. Aim 3 probes a potential connection between Gfi proteins and the regulation of the micro RNA-21 gene locus. Preliminary work indicates that the Gfi proteins occupy chromatin within the miR-21 locus and miR-21 levels are elevated in Gfi-1 deficiency. To test the possible connection in a formal genetic manner in vivo, the requirement for miR-21 in hematopoietic development will be assessed in the context of a conditional mouse mutant at the miR-21 locus, a new mouse strain that is already on hand in this laboratory. If, as suggested, miR-21 participates in control of HSC function or lineage specification, it will provide a new avenue to pursue in development of the blood system. Project Narrative: How blood stem cells and different types of blood cells are programmed at the biochemical and genetic levels is important to our understanding of normal blood cell control, host defense against infection, the properties of adult stem cells, and how cancer is forestalled. The proposed project focuses on mechanisms by which one small family of gene products is critical to blood cell development and function. One long-term outcome of the findings of this work may be improved methods for expansion of blood stem cells, an advance that would facilitate many treatments based on bone marrow transplantation.